Synthesis of epothilones

ABSTRACT

Commercially feasible methods for synthesizing various epothilones precursors needed for the preparation of final epothilones are provided, including techniques for the synthesis of epothilone segment A and C precursors. Segment C precursors are prepared using starting nitriles, which can alternately be oxidized to ketones and converted, or reacted to form the diol with subsequent conversion to the segment. Segment A precursors are prepared by reacting a starting enone with a chiral catalyst to give an intermediate alcohol in high enantomeric excess, followed by conversion of the alcohol to the desired Segment A precursor.

[0001] RELATED APPLICATION

[0002] This is a continuation-in-part of application Ser. No.09/798,196, filed Mar. 2, 2001, which is a continuation of applicationSer. No. 09/280,207, filed Mar. 29, 1999, now U.S. Pat. No. 6,211,412,issued Apr. 3, 2001.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention is broadly concerned with methods forsynthesizing various epothilone segments or precursors (either naturallyoccurring or analogs thereof) which can be used for the efficientsynthesis of complete epothilones.

[0005] 2. Description of the Prior Art

[0006] The epothilones (16-membered macrolides which were initiallyisolated from the myxobacterium Sorangium cellulosum) represent a classof promising anti-tumor agents, and have been found to be potent againstvarious cancer lines, including breast cancer cell lines. These agentshave the same biological mechanism of action as Taxol, an anti-cancerdrug currently used as a primary therapy for the treatment of breastcancer. Other potential applications of the epothilones could be in thetreatment of Alzheimer's disease, malaria and diseases caused bygram-negative organisms. Other cancers such as ovarian, stomach, colon,head and neck and leukemia could also potentially be treated. Theepothilones also may have application in the treatment of arthritis.

[0007] In comparison to Taxol®, the epothilones have the advantage ofbeing active against drug-resistant cell lines. Drug resistance is amajor problem in chemotherapy and agents such as the epothilones haveovercome this problem and hold great promise as effective agents in thefight against cancer.

[0008] In addition, the poor water solubility of Taxol® has led to theformulation of this drug as a 1:1 ethanol-Cremophor concentrate. It hasbeen determined that the various hypersensitive reactions in patientssuch as difficulty in breathing, itchiness of the skin and lowblood-pressure are caused by the oil Cremophor used in the formulation.The epothilones are more water soluble than Taxol® which has positiveimplications in its formulation. Further advantages of the epothilonesinclude easy access to multi-gram quantities through fermentationprocedures. Also the epothilones are synthetically less complex, thusstructural modifications for structure activity relationship studies areeasily accessible.

[0009] The epothilones exhibit their activity by disrupting uncontrolledcell division (mitosis), a characteristic of cancer, by binding toorganelles called microtubules that are essential for this process.Microtubules play an important role in cell replication and disturbingthe dynamics of this component in the cell stops cell reproduction andthe growth of the tumor. Antitumor agents that act on the microtubulecytoskeleton fall into two general groups: (1) a group that inhibitsmicrotubule formation and depolymerizes microtubules and, (2) a groupthat promotes microtubule formation and stabilizes microtubules againstdepolymerization. The epothilones belong to the second group and havedisplayed cytotoxicity and antimitotic activity against various tumorcell lines.

[0010] It has been demonstrated on the basis of in vitro studies thatthe epothilones, especially epothilone B, are much more effective thanTaxol® against the multi-drug resistant cell line KBV-1. Preliminary invivo comparisons with Taxol® in CB-17 SCID mice bearing drug-resistanthuman CCRF-CEM/VBL xenografts have shown that the reduction in tumorsize was substantially greater with epothilone B in comparison toTaxol®.

[0011] In light of the great potential of the epothilones aschemotherapeutic agents, there is a need for techniques allowing thepractical, large scale, economical synthesis thereof. Furthermore, thereis a need for synthetic methods which facilitate the preparation ofvarious homologs and analogs of the known epothilones, and those havingaffinity labels allowing study of the binding interactions of thesemolecules.

SUMMARY OF THE INVENTION

[0012] The present invention overcomes the problems outlined above, andprovides various practical, commercially feasible synthetic routes forthe production of important epothilone precursors or segments in highyield. The invention is particularly concerned with the synthesis of theprecursors or segments C, D (which is a combination of segments B and C)and vinyl halide epothilone precursors.

[0013] In a first aspect of the invention, a C1-C6 segment C epothiloneprecursor of the formula

[0014] Is synthesized using a Noyori reduction reaction. In theforegoing formula, n₁ is an integer from 0-4, R₄ is selected from thegroup consisting of H, C1-C10 straight and branched chain alkyl groups,substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups,R₅ and R₆ are each individually and respectively selected from the groupconsisting of H, substituted and unsubstituted aryl and heterocyclicgroups, C1-C10 straight and branched chain alkyl groups, and substitutedand unsubstituted benzyl groups, R₇ is H or straight or branched chainC1-C10 alkyl groups, and P′ is a protective group (as used herein, P′ isany suitable protective group, and where more than one P′ is in a singleformula, the protective group may be the same or different).

[0015] The method comprises the steps of first providing a β-keto esterof the formula

[0016] Where n₁, R₅, R₆, R₇ and P′ are as defined above, and T is analkyl group. This β-keto ester is then preferentially hydrogenated atthe C3 keto group to form the corresponding hydroxyester. This isaccomplished by reacting the β-keto ester with a hydrogenating agent inthe presence of an asymmetric organometallic molecular catalystcomprising a metal atom or ion having one or more chiral ligands coupledthereto. The synthesis is completed by then converting the hydroxyesterto the epothilone precursor.

[0017] More preferably, n₁is an integer from 0-4, R₅, R₆ and R₇ are eachindividually and respectively selected from the group consisting of Hand the straight and branched chain C1-C4 lower alkyls, and theprotective group is benzyl. In terms of preferred process parameters,the hydrogenating agent is preferably H₂ and the hydrogenating step iscarried out at a pressure of from about 30-100 psi, more preferably50-75 psi, and at a temperature of from about 40-100° C., morepreferably from about 50-75° C. The reaction is normally allowed toproceed for a period of from about 12 hours to 5 days, and more usuallyfor about 2-5 days. Typically, the reaction mixture is agitated duringthe hydrogenating step.

[0018] The catalyst used in the hydrogenation reaction is preferably oneof the well-known Noyori catalysts such as RuBr₂(S)-binap. However, avariety of other catalysts of this type can also be employed. Thecatalyst is generally used at a level of from about 1-25 mol % in thereaction mixture.

[0019] In order to complete the reaction sequence, the hydroxyesterresulting from the Noyori reduction is converted to the epothiloneprecursor segment C. A number of routes can be used to effect thisconversion. Preferably, however, the conversion involves: (1) removingthe P′ protecting group from the hydroxyester to form a diol; (2)protecting the oxygen atoms of the diol, forming a protected diol; (3)reducing the ester function of the protected diol to a primary alcohol;(4) oxidizing the primary alcohol to the corresponding aldehyde; (5)reacting the aldehyde with a Grignard reagent having the R₄ groupcoupled thereto to form a secondary alcohol; and (6) oxidizing thesecondary alcohol to form the final epothilone precursor.

[0020] Preferably, the P′ removal step involves reacting thehydroxyester with hydrogen in the presence of a catalyst (e.g., Pd(OH)₂or Pd/C) at a pressure of from about 40-100 psi. The oxygen atomprotecting step comprises reacting the diol with TBS chloride in acompatible solvent (i.e., one that will not interfere with the desiredreaction) at a temperature of from about 40-100° C. for a period of fromabout 30-60 hours. The ester function reduction step is preferablycarried out by reacting the protected diol with the reducing agentDIBAL-H at a temperature of from about −20 to −85° C. The oxidation ofthe primary alcohol is carried out most conveniently using4-methylmorpholine N-oxide and a catalytic amount of tetrapropylammoniumperruthenate. The Grignard reaction serving to attach the R₄ group isentirely conventional and well within the skill of the art; likewise,the final oxidation of the secondary alcohol is trivial using theaforementioned oxidation procedure, i.e., NMO and TPAP.

[0021] The C1-C6 Formula I segment C can also be produced by a synthesiswherein a nitrile compound of the formula

[0022] Where P′, R₇ and n₁, are as defined above and the value of eachn₁, may be the same or different, is alkylated to yield a dialkylatednitrile compound of the formula

[0023] Where P′, R₅, R₆, R₇, and n₁, are as defined above and the valueof n₁, may be the same or different; and the dialkylated compound isthen converted to the desired C1-C6 segment C epothilone precursor.

[0024] The converting step preferably involves oxidizing the dialkylatedcompound III to yield a ketone of the formula

[0025] Where P′, R₄, R₅, R₆, and n₁, are as defined above and the valueof each n₁, may be the same or different, and converting the ketone tothe C1-C6 epothilone precursor.

[0026] Alternately, the dialkylated nitrile compound defined above maybe treated by deprotecting the nitrile to yield a diol compound havingthe formula

[0027] Where R₅, R₆, R₇ and n₁, are as defined above and the value ofeach n₁, may be the same or different, and thereafter converting thediol compound to the C1-C6 epothilone precursor.

[0028] A still further synthesis of the Formula I C1-C6 segment Cprecursor comprises providing an ester compound of the formula

[0029] Where R₅, R₆, R₇, P′ and n₁, and R′ is a C1-C10 straight orbranched chain alkyl group reacting the ester compound VIII with asulfone to acylate the ester, and thereafter desulfonating the acylatedester to obtain the desired segment C epothilone precursor. The sulfoneis preferably of the formula

X₁—SO ₂—R₄  VIII

[0030] Where R₄ is defined above and X₁ is selected from the groupconsisting of unsubstituted aryl and heterocyclic groups. The mostpreferred sulfone is ethyl phenyl sulfone.

[0031] In another aspect of the invention, a method is provided for theproduction of D precursors, which are a combination of segments B and C.The segment C precursors are of course produced as outlined above.Segment B precursors are of the formula

[0032] Where n₂ is an integer from 1-4, and R₃ is selected from thegroup consisting of H, C1-C10 straight and branched chain alkyl groups,substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups.This segment can be efficiently produced using known techniques.

[0033] The segments B and C are connected by first reacting the segmentC precursor with a base to form an enolate, followed by reacting theenolate with the segment B. These reactions are generally carried out byinitially cooling the base to a temperature of about −75° C., adding thesegment C precursor and elevating the temperature of the mixture toabout −40° C., then recooling the mixture to at least about −75° C. andadding the precursor segment B thereto.

[0034] The invention also is concerned with a method of synthesizingvinyl halide epothilone precursors having the general formula

[0035] Where n₃ is an integer from 1-4, R is selected from the groupconsisting of C4-C8 cycloalkyl, and substituted and unsubstitutedaromatic and heteroaromatic groups, R₁ and R₂ are each individually andrespectively selected from the group consisting of H, C1-C10 straightand branched chain alkyl groups, substituted and unsubstituted benzylgroups, and C1-C10 alkoxy groups, P′ is a protecting group, and M iseither bromine or iodine. This reaction involves first providing analkynyl ketone of the formula

[0036] Wherein n₃ and P′ are as previously defined. Thereafter, thealkynyl ketone is asymmetrically reduced to create the alcohol form ofthe alkynyl ketone. This alcohol form is then reacted with a reagentsystem selected from the group consisting of (R₁)₃Al and zirconocenedichloride or stannyl cupration reagent and R₁-halide to form a vinylmetal species. The vinyl metal species is then reacted with an aryl orvinyl halide to form an allyl alcohol. This allyl alcohol is thenconverted to the vinyl halide epothilone precursor.

[0037] Normally, the asymmetric reduction step involves creating thereduced form of the alkynyl ketone and the resulting alcohol isprotected using TBS as a protecting agent. The R₁-halide is selectedfrom the group consisting of R₁Br and R₁I. The conversion steppreferably includes the step of initially converting the allyl alcoholto an alkynyl stannane, reducing the stannane withchlorohydridozirconocene to form a 1,1-dimetallo Zr-Sn species. Thedimetallo species is then hydrated to form a vinyl stannane, which isthen quenched with either iodine or bromine. Alternately, the conversionstep may be accomplished by transmetallating the dimetallo species withan organocuprate, quenching with an alkyl-R₂-OTf, and final quenchingwith either iodine or bromine incorporating the R₂ group.

[0038] Preferred vinyl halide C12-C20 epothilone precursors of theformula

[0039] Where R₈ is selected from the group consisting of H, C1-C4straight or branched chain alkyl, alkenyl or alkynyl groups, R₉ isselected from the group consisting of H, C1-C10 straight and branchedchain alkyl, alkenyl, alkynyl, hydroxyalkyl, hydroxyalkenyl orhydroxyalkynyl groups, substituted and unsubstituted cyclic, heteroxylicand aryl groups, R₁₂ is selected from the group consisting of H, C1-C10straight and branched chain alkyl groups, substituted and unsubstitutedbenzyl groups, and C1-C10 alkoxy groups, X₂ is O or S, n₄ is an integerwhich ranges from 1 to 4, P′ is a protective group and M is eitheriodine or bromine, may be produced as follows.

[0040] First, an alcohol of the formula

[0041] Where R₈, R₉, X₂, n₄ and P′ are as defined above, is converted tothe C12-C20 epothilone segment A. This method preferably comprises thesteps of:

[0042] providing an enone compound of the formula

[0043] Where R₈, R₉, X₂, P′ and n₄ are as defined above, andasymmetrically reducing the enone compound XIV in the presence of achiral catalyst to obtain the alcohol, compound XIII. The alcoholcompound XIII is then protected at the C15 alcohol position, followed byknown conversion steps to precursor Formula XII.

[0044] The enone compound XIV is preferably obtained by reacting in abasic reactive medium starting aldehyde compound of the formula

[0045] Where R₉ and X₂ are as defined above, with a phosphonate compoundof the formula

[0046] Where R₈, P′ and n₄ are as defined above, and R₁₀ and R₁₁ areindividually selected from the group consisting of C1-C4 straight orbranched chain alkyl groups. In particularly preferred forms, R₈ and R₉are each H, X is S, n₄ is 1, and P′ is TBS. The chiral catalyst ispreferably (R)-B-Me-CBS-oxazaborolidine.

[0047] A still further method of synthesizing the preferred C12-C20epothilone precursors of Formula XII described immediately aboveinvolves conducting an aldol condensation reaction using an aldehydewith an enolate anion to give a β-keto alcohol; this alcohol is thenoxidized to the ester form followed by an asymmetric reduction to yielda chiral alcohol. Preferably, the method comprises providing an aldehydeof the formula

[0048] Where R₈, R₉ and X₂ are as defined above, reacting this aldehydewith an acetate of the formula

[0049] Where R₁₃ is a methyl group, Z is a C1-C4 straight or branchedchain alkyl group or a substituted or unsubstituted benzyl group in abasic reaction mixture to yield a β-hydroxyester of the formula

[0050] Where R₈, R₉, X₂, and Z are as defined above.

[0051] The β-hydroxyester is then oxidized to the correspondingβ-ketoester of the formula

[0052] Where R₈, R₉, X₂, and Z are as defined above. Next, β-ketoesteris hydrogenated to form a chiral alcohol of the formula

[0053] Where R₈, R₉, X₂, and Z are as defined above, by reacting theβ-ketoester with a hydrogenating agent in the presence of asymmetricorganometallic molecular catalyst comprising a metal atom or ion havingone or more chiral ligands coupled thereto. Finally, the chiral alcoholis converted to the C12-C20 epothilone of Formula XII.

[0054] In preferred forms, the acetate is ethyl acetate, and thealdehyde and acetate are reacted in the presence of an alkali metaldiisopropyl amide in a solvent selected from the group consisting ofTHF, a mixture of t-butanol and t-butoxide, sodium ethoxide, andethanol. The reaction temperature is preferably from about −50 to −125°C. The β-ketohydroxyester is preferably oxidized using an alkali metalor alkaline earth metal oxide or hydroxide. The hydrogenating steppreferably uses hydrogen and is carried out at a pressure of from about30-100 psi, and a temperature from about 40-100° C.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0055] The molecular architecture of the representative epothilones(Formulae A-B) reveals three essential domains. These include the twochiral domains, namely the C1-C8 polypropionate region and the C12-C15region, and the achiral spacer C9-C11 which unites the chiral domains.Additional structural features include a thiazole moiety, the C16 doublebond, a methyl group at C4 and a cis-epoxide moiety (C12-C13) in theepothilones of Formula A. In the following formulae A and B, n, is aninteger from 0-4, n₂ and n₃ are each respectively integers from 1-4, Ris selected from the group consisting of C4-C8 cycloalkyl, andsubstituted and unsubstituted aromatic and heteroaromatic groups, R₁,R₂, R₃ and R₄ are each individually and respectively selected from thegroup consisting of H, C1-C10 straight and branched chain alkyl groups,substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups,R₅ and R₆ are each individually and respectively selected from the groupconsisting of H, substituted and unsubstituted aryl and heterocyclicgroups, C1-C10 straight and branched chain alkyl groups, and substitutedand unsubstituted benzyl groups, R₇ is H, or straight or branched chainC1-C10 alkyl groups, X is either oxygen or NH, and Y is either oxygen orH₂.

[0056] Scheme 1 below outlines a retrosynthetic analysis respecting thetotal synthesis of the epothilones of Formula A in accordance with theinvention, where each n₁, and n₂ equal 1, R is 2-methyl-thiazol-4-yl,R₁, is methyl, R₂ is H or methyl, R₃, R₄, R₅ and R₆ are methyl, R₇ is H,and X and Y are oxygen. Standard epoxidation and macrolactonizationstrategies are used for the formation of the C12-C13 epoxide moiety andthe 16-membered macrolide. The analysis for other analog epothilones ofFormula A is identical, and also for the epothilones of Formula B andits analogs, with the epoxidation step being omitted.

[0057] A novel route to a Formula I C1-C6 segment (labeled C inScheme 1) utilizes a stereoselective hydrogenation reaction, i.e., aNoyori reduction.

[0058] Synthesis of Segment C (C1-C6 of Formula A)

[0059] The invention makes it possible to synthesize several analogs ofsegment C as set forth in Formula I with various chain elongationsand/or substitutions at C2 and substitutions at the α-carbon relative tothe keto group. It also allows for, as mentioned before, modificationsat the carbon atom between the keto and the protected secondary hydroxygroup with other groups. These chain extensions and substitutions areillustrated by a general Formula I, previously identified. The synthesisof these modified derivatives can be achieved utilizing chemistryexemplified in the synthesis of segment C in the Schemes describedbelow. These modified segments can then be utilized in the totalsynthesis of various analogs of epothilones.

[0060] The synthesis of the Forumula I segment has been accomplished viaunique and complementary routes, detailed in Schemes 2 and 3 below,which illustrates the synthesis of the naturally occurring segment C. Anovel step in the synthesis of the C1-C6 segment utilizes the Noyorihydrogenation of β-keto ester 4 to generate the requisitestereochemistry at C3. This Noyori hydrogenation (Noyori, R. et al.,Asymmetric Hydrogenation of, β-Keto Carboxylic Esters. A Practical,Purely Chemical Access to β-Hydroxy Esters in High Enantiomeric Excess,J. Am. Chem. Soc. 109:5856-5858 (1987)) provides the required enantiomerwith high selectivities (92-95% enantiomeric excess). The use of aNoyori hydrogenation reaction permits large, commercial scale productionof various segment C precursors.

[0061] The required β-keto ester 4 is obtained in two steps from thereadily available starting material 3-benzyloxypropionic acid (2).Asymmetric hydrogenation of 4 in methanol using RuBr₂(S)-binap ascatalyst at 60 psi gives the β-hydroxyester 5 in 71-92% yield (92-95%ee). Deprotection of the benzyl ether and bis-silylation of theresultant diol 6 provides ester 7. The ester is reduced to the knownprimary alcohol 8 using DIBAL-H. The alcohol is then oxidized to theknown aldehyde 9 using a previously unreported oxidation procedure. Thealdehyde is then reacted with EtMgBr using a reported procedure (Claus,et al., Synthesis of the C1-C9 Segment of Epothilons, Tetrahedron Lett.,38:1359-1362 (1997)) to give the known secondary alcohol 10 in 65%yield. This alcohol is then oxidized to the C1-C6 segment C using TPAPand NMO.

[0062] In summary, although segment C is a key synthon in previouslyreported total syntheses (Nicolaou, et al., Total Syntheses ofEpothilones A and B via a Macrolactonization-Based Strategy, J. Am.Chem. Soc., 119:7974-7991 (1997)) of the epothilones, the syntheticroute utilizing the asymmetric Noyori hydrogenation is unique.

[0063] The alternate route toward segment C precursors allows for theintroduction of affinity labels and modifications at the C4 position asshown in Scheme 3. Applying the Noyori reduction to the knownunsubstituted β-keto ester 11 provides a building block that can be usedfor the modifications at C4 of the epothilones. This Scheme accordinglyallows for modification of the epothilones and gives a more generalroute to introduce a variety of substituents at this position.

[0064] Thus, the Noyori hydrogenation of β-keto ester 11 yields theknown β-hydroxy ester 12 (Ali, et al., Formal Syntheses of Cryptophycin1 and Arenastatin A, Tetrahedron Lett., 38:1703-1706 (1997)) in 97%yield (in 97% enantiomeric excess). The Frater alkylation of β-hydroxyester 12 yields the previously reported α-methyl analogue 13 (Ali, etal., Formal Syntheses of Cryptophycin 1 and Arenastatin A, TetrahedronLett., 38:1703-1706 (1997)) in 71% yield (98% diastereomeric excess). Asecond Frater alkylation of hydroxyester 13 gave bis-dimethyl derivative5 in 59% yield which was then converted to epothilone segment C by thechemistry shown in Scheme 2. At this stage, other substituents such asbenzyl, allyl and other C1-C6 alkyl groups can be introduced by usingother electrophiles in the second Frater alkylation in place ofiodomethane. The novel aspect about this alternate route to segment C isthe ability to alter the substituents at the C4 position of theepothilones using the aforementioned Frater alkylation strategy.

[0065] In another aspect of the invention, the synthesis of exemplarysegment C (and of course all of the other segment C analog precursors ofFormula I) utilizes a starting material which can be obtained fromlactose or malic acid and circumvents the need to construct the C3stereochemistry using an asymmetric synthesis. This technique givesaccess to the C1-C6 segment of the epothilones by a concise route setforth in Scheme 3A.

[0066] The Scheme 3A synthesis employsethyl-(R)-4-cyano-3-hydroxybutanoate 12 as starting material. Selectivereduction of the ester functionality using sodium borohydride in ethanolfrom 0° C. to room temperature overnight gave(S)-3,5-dihydroxyvaleronitrile 13.

[0067] The product 13 was then protected at its free 4-hydroxyl group asa p-methoxybenzyl ether by forming a dibutyl tin acetal with dibutyltindimethoxide in refluxing benzene, followed by treatment withp-methoxybenzyl chloride and tetrabutylammonium iodide at 60° C. to givethe primary ether derivative 14 in 61% yield.

[0068] The nitrile 14 is then alkylated using LDA and methyl iodide. Theenolate of the nitrile generated using LDA is warmed to 60° C. beforethe addition of methyl iodide to ensure dialkylation to give(S)-2,2-dimethyl-3-hydroxy-5-p-methoxybenzyloxy-valeronitrile 15 in 76%yield.

[0069] The dialkylated product 15 thus obtained is then refluxed withethyl magnesium bromide in a THF solution with a catalytic quantity ofcopper bromide-dimethylsulfide complex and the resulting iminehydrolyzed in situ with 0.5 N aqueous citric acid solution for 5 hrs togive ketone 16. Deprotection of the PMB group on the ketone 16 withceric ammonium nitrate with a 1:9 water:acetonitrile solvent mixturefollowed by protection of the diol with TBSOTf and 2,6-lutidine gave theketone 18 which constitutes segment C, the C1-C6 carbon skeleton of theepothilones.

[0070] An even more preferred synthesis of the C1-C6 segment Cprecursors is a two-step, one-pot conversion of an intermediatemethylester to the ethyl ketone using a sulfone anion to acylate theester, followed by desulfonylation to provide segment C using sodiumamalgam. This one-pot conversion achieves 90% yield and shortens thesynthesis significantly. This preferred synthesis is set forth in thefollowing Scheme 3B; again this scheme may be readily modified to obtaindesired analogs defined by Formula I.

[0071] Synthesis of Segment B (C7-11 of Formula A)

[0072] The synthesis of the C7-C11 segment B is preferably achievedusing previously reported chemistry (Lin, Efficient Total Syntheses ofPumiliotoxins A and B. Applications of Iodide-Promoted IminiumIon-Alkyne Cyclization in Alkaloid Construction, J. Am. Chem. Soc.,118:9062-9072(1996)) and is outlined in exemplary Scheme 4, which isprecursor of a naturally occurring epothilone.

[0073] This synthesis can also be used to introduce variouschain-elongations on this segment and to introduce various othersubstituents at C-8. These modifications can be illustrated by FormulaIX (Segment D), wherein n₂ and R₃ are as defined previously. Theirsynthesis can be achieved using chemistry exemplified in the synthesisof segment B in Scheme 4. Again, these modified segments can then beutilized in the total synthesis of various analogs of epothilones.

[0074] Synthesis of Segment D (C1-C11 of Formula A) via Aldol Reaction

[0075] The connection of the two segments C and B utilizes a highlydiastereoselective aldol reaction, exemplified in Scheme 5 showing theconnection of the two precursors B and C of a naturally occurringepothilone. When the C1-C6 ketone segment C is treated with abase, forexample lithium diisopropylamide and the resultant enolate reacted withC7-C11 aldehyde segment B, a single desired diastereomer 14 was observedin 65% yield. This diastereselectivity is believed to arise from afavorable nonbonding interaction between the C10-C11 double bond and thecarbonyl group of the aldehyde that gives rise to the desireddiastereomer. After the connection is made, the resultant secondaryalcohol is protected as the corresponding tert-butyldimethylsilyl ether.

[0076] Similar chemistries would apply for the connection of modifiedsegments C and B of the type discussed previously and emplified byFormulae C and D.

[0077] Proposed Synthesis of Segment A (C12-C20 of Formula A)

[0078] The invention also provides a new route to the C12-C20 segment(segment A of the naturally occurring epothilone), and correspondinganalogs thereof. This involves new ways to set the C16-C17trisubstituted double bond and the C12-C13 cis-double bond, which servesas precursor to the cis-epoxide at C12-C13 in the epothilones.

[0079] Stereoselective Construction of C16-C17 of Trisubstituted Olefinand Introduction of Thiazole in Formula A

[0080] The introduction of the thiazole moiety draws uponzirconium-catalyzed carboalumination chemistry (Wipf, RapidCarboalumination of Alkynes in the Presence of Water, Agnew. Chem., Int.Ed. Engl., 32:1068-1071 (1993)) wherein a C16-C17 alkyne bond in anappropriately functionalized C13-C17 propargylic alcohol 16 (Scheme 6)is subjected to methylalumination in the presence of zirconocenedichloride (Cp₂ZrCl₂). The resultant alkenylalane is coupled with2-methyl-4-bromothiazole 17 in the presence of zinc chloride under Pd(0)catalysis to access the trisubstituted E-olefin 19 stereoselectivelyfollowing the protection of the alcohol 18 as the OTBS-ether.

[0081] The chiral propargylic alcohol 16 is obtained via the asymmetricreduction of the readily available alkynyl ketone 15. This isexemplified in Scheme 6, which illustrates the synthesis of theprecursor for the naturally occurring epothilone. After the introductionof the thiazole moiety, the known primary alcohol 21 is revealed bydeprotection of the PMB ether 19 and then oxidized to the previouslyreported (Mulzer, J., et al. Easy Access to the EpothiloneFamily—Synthesis of Epothilone B, Tetrahedron Lett., 39:8633-8636(1998)), C13-C20 aldehyde 22.

[0082] Alternately, a stannylcupration-methylation methodology (Harris,et al., Synthetic Approaches to Rapamycin. 3. Synthesis of a C1-C21Fragment, Synlett, pp. 903-905 (1996)) can be used in order to introducethe trisubstituted olefin. Thus the O-TBS ether 16a (Scheme 7) ofpropargylic alcohol 16 on treatment with the stannylcuprate reagent 20followed by methylation with iodomethane provides the correspondingstannane which is then coupled under Stille conditions with thebromothiazole 17 to yield the olefin 19.

[0083] The synthesis of 2-methyl-4-bromothiazole 17 from the known2,4-dibromothiazole (Reynaud, et al., Sur une Nouvelle Synthese du CycleThiazolique, Bull. Soc. Chim. Fr., 295:1735-1738( 1962)) is outlined inScheme 8.

[0084] The zirconium-catalyzed methylalumination strategy constitutes anovel route to construct the C16-C17 double bond and to introduce thethiazole ring. The novelty lies in the use of a chiral propargylicalcohol like 16 in the carbometalation reaction followed by the directintroduction of the thiazole unit.

[0085] This methodology also allows for the introduction of varioussubstituents and chain elongations on the C12-C20 segment A. Thusstarting with analogs of the ketone 15 in Scheme 6, a variety ofchain-elongated derivatives of segment A can be produced. Also carryingout an ethylalumination (Et₃Al) in place of methylalumination (AlMe₃)(Scheme 6) allows the introduction of an ethyl group (Et) at C16. In thesame context, other groups can also be introduced using the alternatestannylcupration-alkylation method by replacing iodomethane with otherelectrophiles in this reaction shown in Scheme 7. In addition, thethiazole ring can be replaced by other cyclic, aromatic andheteroaromatic rings by using other vinyl or aromatic/heteroaromatichalides in place of 2-methyl-4-bromothiazole 17 in the coupling reactionfollowing either the carboalumination or stannylcupration strategyexemplified in Schemes 6 and 7 respectively.

[0086] Stereoselective Construction of the C12-C13 Cis-olefinic Bond ofFormula A

[0087] The goals in the construction of the C12-C13 Z-olefinic bond,were to design a method providing maximum control over the olefingeometry and to furnish common intermediates in the synthesis of bothepothilones A and B. The introduction of affinity labels at C-12 wasalso a consideration.

[0088] The C12-C13 olefin can be constructed in the form of Z-vinyliodides I that can be obtained from vinylstannanes with definedconfigurations. The vinyl stannanes will be accessed by using knownchemistry reported by Lipshutz et al., Preparation of Z-Vinylstannanesvia Hydrozirconation of Stannylacetylenes, Tetrahedron Lett.,33:5861-5864 (1992); Lipshutz, et al., Hydrozirconation/Transmetalationof Acetylenic Stannanes. New 1,1-Dimetallo Reagents, Inorganica ChimicaActa, 220:41-44 (1994), which utilizes a 1,1-dimetallo species as astereodefined 1,1-vinyl dianion synthon. An exemplary synthesis is givenin Scheme 9, for the precursor to a naturally occurring epothilone, andstarts with a Corey-Fuchs reaction (PPh₃, CBr₄) of the known aldehyde22, followed by base-induced elimination and quenching of the lithiumacetylide with tributyltin chloride (Bu₃SnCl) to yield alkynylstannane23. The 1,1-dimetallo species 24 is generated by hydrozirconation of thealkynyl stannane 23 using chlorohydridozirconocene (Schwartz reagent).An aqueous quench would provide Z-vinylstannane 25a or alternatively,selective transmetalation with a higher order cuprate, followed byaddition of an electrophile (MeOTf in case of epothilone B) to theresultant species provides the a-substituted vinylstannane 25b with highstereoselectivity. The Z-vinylstannanes 25a and 25b can then betransformed to the corresponding vinyl iodides I utilizing iodine withretention of configuration.

[0089] An alternative route to the synthesis of alkynylstannane 23(Scheme 9a) which allows for incorporation of different substituents atthe C16 carbon involves the asymmetric epoxidation of secondary alcohol18a under the Sharpless conditions using (−)-diisopropyl tartrate,tert-butyl hydroperoxide and titanium isopropoxide to give epoxide 19a.The alcohol function on the epoxide can be oxidized with TPAP, NMO togive ketone 20a which can be reacted with Wittig reagents containingthiazole or other aromatic/heteroaromatic rings to give thecorresponding trans-olefins. The terminal epoxide in this olefin canthen be opened with trimethylsilyl acetylide to give secondary alcohol22a. The trimethylsilyl group can then be substituted for a trialkylstannyl group on treatment of 22a with TBAF and bis-tributyltin oxideand the obtained product treated with TBSCl to give compound 23.

[0090] The foregoing chemistries can be used for the synthesis of analogprecursors as well. Such analogs are best illustrated by Formula X,wherein n₃, R, R₁ and R₂ are as defined previously. Again all of thesemodified segments can then be utilized in the total synthesis of variousanalogs of epothilones.

[0091] In summary, although some of the vinyl iodides of the Formula Xare previously reported (20,21) compounds, the method to synthesize itfrom the known aldehyde 22 is different from conditions reported inother total syntheses of epothilones. In addition, the above mentionedhydrozirconation reactions provide precise control over the geometry ofthe C12-C13 olefin bond. Also the use of other electrophiles in thetransmetalation reaction with the intermediate species 24 allows for thesynthesis of various analogs.

[0092] The invention also provides new synthetic routes to specificpreferred embodiments of the above Formula X defined previously, inparticular C12-C20 vinyl halide epothilone precursors of the formula.

[0093] A preferred reaction Scheme 9B set forth below illustrates thisaspect of the invention. Thus, the known aldehyde 1 was treated with thephosphonate 2 in presence of barium hydroxide and wet tetrahydrofuran assolvent to give the enone 3 in 75% yield. The asymmetric reduction ofthis enone 3 using 50 mol % of commercially available chiral catalyst(R)-2-Me-CBS-oxazaborolidine and 1.5 equivalents of boranedimethylsulfide complex in dichloromethane gave the desired alcohol 4 in79% yield and in 95% enantiomeric excess. The completion of the segmentsynthesis involved the protection of the C15 alcohol as the TBS ether toprovide the known compound 5 using TBSOTf (tert-butyldimethylsilyltrifluoromethanesulfonate) and 2,6-lutidine as base. The remaining steps(i.e.conversion of 5 to I) to the known vinyl iodide I have beenpreviously reported in literature.

[0094] An important feature of this synthesis is the ability to producein high enantomeric excess the alcohol 4 from the enone 3, using achiral catalyst. This largely eliminates racemates in the alcohol, thusgiving significantly higher yields.

[0095] A second method to the same C12-C20 precursor relies on the useof a Noyori asymmetric hydrogenation of β-ketoesters to set the requiredstereochemistry for the C15 stereocenter of the epothilones. Thischemistry is illustrated in Scheme 9C below, and begins with the knownaldehyde 6. An aldol condensation reaction of this aldehyde 6 with theenolate anion generated from ethyl acetate using lithium diisopropylamide as base provided the beta-ketoalcohol 7 in 76% yield. This alcohol7 was oxidized to the beta-ketoester 8 using MnO₂ (manganese dioxide) in90% yield. The Noyori asymmetric reduction of the beta-ketoester 8provides the chiral alcohol 9 with desired S-stereochemistry at position15 in 50% yield and 83% enantiomeric excess. This alcohol 9 is protectedas its TBS (tert-butyldimethylsilyl) ether 10 using TBSOTf(tert-butyldimethylsilyl trifluoromethanesulfonate) and 2,6-lutidine in72% yield. The ester functionality in 10 was then reduced to the knownprimary alcohol 11 using DIBAL-H. The final stages in the synthesis aresimilar to those reported in Scheme 1 and the same conversions have beenreported previously in the literature.

[0096] Two other epothilone derivatives of special interest maybesynthesized in accordance with the invention. In one such derivative thelactone functional group is replaced with an ether functionality and inthe other a lactam functionality is used in lieu of the lactonefunctional group. Thus in the first derivative, and referring to FormulaA, X is O, Y is H₂, n₁, n₂, and n₃ are 1, R is 2-methyl-thiazol-4-yl, R₁is methyl, R₂ is H or methyl, and R₃, R₄, R₅ and R₆ are methyl. In thesecond derivative, the only change is that X is NH and Y is O. Thesecould be synthesized by the reaction sequences shown in Schemes 10 and11. Thus selective deprotection at C1 by camphorsulfonic acid (CSA)(Scheme 10), formation of the mesylate derivative of the correspondingprimary alcohol, selective deprotection of the C15 TBS ether andbase-induced cyclic ether formation should provide compounds 26′. Again,the final stages in the synthesis would involve the deprotection of boththe TBS groups from the macrolides (TFA, CH₂Cl₂) and thediastereoselective epoxidation of the C12-C13 double bond withepoxidizing agents such as dimethyldioxirane to give the etherderivatives 29 and 30.

[0097] For the lactam formation (Scheme 11) again compound 26 could beselectively deprotected at C-1 followed by sequential oxidation of theprimary alcohol first under Swem conditions followed by NaClO₂—NaH₂PO₄would furnish the known acids. These known acids can be converted totheir allyl esters and then the TBS ether at C15 can be deprotectedselectively. Mitsunobu inversion of these alcohols and azide formationvia the corresponding mesylates will provide the azides with the correctstereochemistry at C15. Reduction of the azides (PPh₃, H₂O) followed bysalt formation of the amine will provide 32. Deprotection of the allylesters (Pd(PPh₃)₄, base) followed by macrolactamization (HBTU) willprovide the lactams 33. Again, deprotection of both the TBS groups fromthe macrolides (TFA, CH₂Cl₂) and the diastereoselective epoxidation ofthe C12-C13 double bond with epoxidizing agents such asdimethyldioxirane would give the lactam derivatives 34 and 35.

[0098] Representative C4-C8 cycloalkyl, substituted and unsubstitutedaromatic and heteroaromatic groups, C1-C10 straight and branched chainalkyl groups, substituted and unsubstituted benzyl groups, C1-C10 alkoxygroups, and heterocyclic groups useful in the formation of epothiloneanalogs are set forth below.

[0099] C4-C8 cycloalkyl groups: cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl.

[0100] Substituted and unsubstituted aromatic groups: phenyl, phenylgroups substituted at any position with C1-C4 straight or branched chainalkyls, C1-C4 alkoxy groups, halogens, amines, amides, azides, sulfides,carboxylic acids and their derivatives, and hydroxides.

[0101] Substituted and unsubstituted heteroaromatic groups: thiazoles,pyrroles, furans, thiophenes, oxazoles and pyridines, and imidazoles.

[0102] C1-C10 straight and branched chain alkyl groups: methyl, ethyl,propyl, butyl, isopropyl, isobutyl, isopentyl, octyl, nonyl, andt-butyl.

[0103] Substituted and unsubstituted benzyl groups: benzyl, benzylgroups substituted at any position with C1-C4 straight or branched chainalkyls, C1-C4 alkoxy groups, halogens, amines, amides, azides, sulfides,carboxylic acids and their derivatives, and hydroxides.

[0104] C1-C10 alkoxy groups: methoxy, ethoxy, propoxy, butoxy,isopropoxy, t-butoxy, and nonoxy.

[0105] Heterocyclic groups: piperidines, furans, pyrroles, oxazolines,and thiophenes.

[0106] The following examples set forth various syntheses of the typedescribed previously. It is to be understood, however, that theseexamples are provided by way of illustration and nothing therein shouldbe taken as a limitation upon the overall scope of the invention.

EXAMPLE 1

[0107] The synthesis of a segment C precursor was accomplished via twounique and complementary routes which are detailed in Schemes 2 and 3.One novel step in the synthesis of the C1-C6 segment utilizes the Noyorihydrogenation as detailed in Noyori, R. et al., Asymmetric Hydrogenationof β-Keto Carboxylic Esters. A Practical, Purely Chemical Access toβ-HydroxyEsters in High Enantiomeric Excess, J.Am. Chem. Soc.,109:5856-5858 (1987). The Noyori hydrogenation of β-keto ester 4 inScheme 2 generates the requisite stereochemistry at C3. This Noyorihydrogenation provides the required enantiomer with high selectivities(95% enantiomeric excess). It is a versatile reaction and has foundnumerous applications in the synthesis of biologically active naturalproducts and is also amenable to large scale synthesis.

[0108] The following is a detail of the procedures that are outlined inScheme 2. The required β-keto ester 4 is obtained in two steps from thereadily available starting material 3-benzyloxypropionic acid (2) asdescribed by Davis et al., Nonracemic α-fluoro aldehydes: Asymmetricsynthesis of 4-deoxy-4-fluoro-d-arabinopyranose, J. Org. Chem.,62:7546-7547 (1997), the teachings of which are hereby incorporated byreference. Isopropylcyclohexyl-amine (7.3 mL, 44.6 mmol, 1.5 eq.) wasdissolved in 40 mL THF. The temperature was then lowered to −30° C. andn-butyllithium (16.1 mL, 38.6 mmol, 1.3 eq.) was added dropwise andstirred for 30 minutes. Next, the temperature was raised to 0° C. for 15minutes and then cooled to −78° C. for 15 minutes. Methyl isobutyrate(3.75 mL 32.7 mmol, 1.1 eq.) was dissolved in 5 mL THF and addeddropwise. The resulting mixture was stirred 30 minutes.3-Benzyloxypropionyl chloride (5.0 g, 29.7 mmol, 1 eq) in 5 mL THF wasthen added dropwise. This reaction mixture was stirred for one houruntil the starting material completely disappears by thin layerchromatography (TLC) (80:20 hexanes/EtOAc). The reaction mixture wasthen quenched with 20 mL 20% HCl and raised to room temperature. Next,the reaction mixture was extracted 3 times with ether and the combinedorganic phases were washed twice with sodium bicarbonate and once withbrine. The combined aqueous layers were cross-extracted twice withether. The organics were combined, dried with Na₂SO₄, and concentratedunder reduced pressure. Purification was achieved via columnchromatography on silica gel using a hexane/EtOAc gradient whichresulted in 5.1 g (65% yield) of β-keto ester 4.

[0109] Asymmetric hydrogenation of β-keto ester 4 in methanol usingRuBr₂(S)-binap as catalyst at 65 psi gave the β-hydroxyester 5 in 85%yield. This was done by the following process. Acetone and MeOH weredistilled and stored over molecular sieves. Each was degassed five timesusing the freeze-thaw method and placed under argon. Noyori's rutheniumcatalyst (91 mg, 0.284 mmol, 1 eq.), preferablybis-(2-methylallyl)cycloocta-1.5-diene ruthenium (II) and (S)-BINAP(S)-(−)-1,1′bi-2-naphthal(s)-(−)-2,2′-Bis(diphenylphosphino)1,1′binaphthyl (177 mg, 0.284 mmol, 1 eq.) werecombined in a Schlenk flask with 24 mL acetone and 2.0 mL HBr solution(0.25 mL 48% HBr, 5.1 mL acetone). The resulting mixture was stirred for4 hours to allow the catalyst to form. The acetone was then removedunder reduced pressure. Next, beta ketoester 4 (5.36 g, 20.3 mmol, 71.5eq.) in 23 mL MeOH was degassed four times and then transferred to aParr hydrogenation flask using MeOH. The catalyst was then rinsed intothe Parr flask using MeOH. The hydrogenation reaction was conducted over110 hours at 65 psi and 60° C. The contents were concentrated underreduced pressure and then taken up in ether. The reaction mixture wasfiltered twice to remove the catalyst and then concentrated. Finalpurification was obtained through column chromatography wherein thecolumn contained silica gel and utilized a hexane/EtOAc gradient andyielded 4.57 g (85% yield).

[0110] Deprotection of the benzyl ether and bis-silylation of diol 6provided ester 7. To produce diol 6, β-hydroxyester 5 (1.824 g, 6.86mmol, 1 eq.) was dissolved in 20 mL THF and transferred to a Parrhydrogenation vessel under argon. Pd(OH)₂ (450 mg, 0.25 eq.) was addedand the flask purged for an additional 10 minutes with argon. Thehydrogenation reaction was conducted at 50 psi for 24 hours. Finally,the reaction mixture was washed through a frit, an ultrafine strainer ina filtration technique preferably fit with 300 mL EtOAc and concentratedunder reduced pressure to give 1.18 g (90% yield) of diol 6.

[0111] In detail, ester 7 was made by taking diol 6 (450 mg, 2.84 mmol,1 eq) dissolved in 3.15 mL DMF and adding imidazole (1.16 g, 17.04 mmol,6 eq) which was stirred until dissolved. TBSCl (tert-Butyldimethylsilylchloride) (1.28 g, 8.52 mmol, 3 eq) was added and the temperature wasraised to 60° C. This reaction mixture was then stirred for 44 hours.Disappearance of the starting material was monitored by thin layerchromatography. The reaction was then quenched with H₂O and NH₄Cl. Afterquenching, the reaction mixture was extracted twice with ether. Theether layer was washed with NaHCO₃ and brine, dried with Na₂SO₄, andconcentrated. Purification via column chromatography (SiO₂, 95:5hexane/EtOAC) gave 0.89 g or a 78% yield.

[0112] Primary alcohol 8 is the result of reducing ester 7 (1.89 g, 4.68mmol, 1 eq) by dissolving it in 26 mL of CH₂Cl₂, cooling it to −78° C.and adding DIBAL-H (9.4 mL, 14 mmol, 1.5M in hexanes) dropwise. Afterstirring at this temperature (−78 ° C.) for one hour, the reaction wasquenched with 10 mL of MeOH. Next, the reaction mixture was warmed to 0°C. and 10 mL of a saturated aqueous solution of potassium sodiumtartarate was added. After stirring this mixture for 16 hours, theaqueous layer was extracted four times with CH₂Cl₂. The combinedorganics were dried over anhydrous sodium sulfate and concentrated toyield 1.59 g (92% yield) of the primary alcohol. Although this is aknown alcohol, the synthetic route from this ester is novel.

[0113] Primary alcohol 8 was oxidized to aldehyde 9 by taking primaryalcohol 8 (1.45 g, 3.86 mmol, 1 eq) and dissolving it in 25 mL CH₂Cl₂.Molecular sieves (4 Å, powdered) were added to aid in the removal ofwater and this mixture was stirred for 15 minutes. 4-methylmorpholineN-oxide (NMO) (0.77 g, 6.56 mmol, 1.7 eq) was then added and afterstirring for 30 minutes, tetrapropylammonium perruthenate (TPAP) (0.081g, 0.23 mmol, 0.06 eq) was added. The reaction mixture was stirred for16 hours at room temperature and then concentrated. It was then passedthrough a pad of 4:1 silica gel:Celite mixture (35 g) to yield 1.2 g(83% yield) of the aldehyde.

[0114] The aldehyde 9 is then reacted with EtMgBr using the procedure ofClaus, E. et al., Synthesis of the C1-C9 Segment of Epothilons, 38Tetrahedron Lett., 1359-1362 (1997), the procedure of which is herebyincorporated by reference, to give the known secondary alcohol 10 in 65%yield.

[0115] This secondary alcohol 10 is then oxidized to the C1-C6 segment Cusing the same procedure that was used to oxidize primary alcohol 8 toaldehyde 9. The alcohol 10 (50 mg, 0.124 mmol, 1 eq) was dissolved in 1mL CH₂Cl₂. Molecular sieves (4 Å, powdered) were added and this mixturewas stirred for 15 minutes. NMO (25 g, 0.211 mmol, 1.7 eq) was thenadded and after stirring for 30 minutes, TPAP (3 mg, 0.0074 mmol, 0.06eq) was added. The reaction mixture was stirred for 15 hours at roomtemperature and then concentrated. It was then purified bypassing itthrough a column (20 g) of 5:1 silica gel:Celite mixture (5% EtOAC inhexane) to yield 46 mg (92%) of the ketone (segment C). Again althoughsegment C is known the oxidation process used is different fromconditions reported.

[0116] In summary, although segment C is a key synthon in previouslyreported total syntheses of the epothilones, the synthetic routeutilizing the asymmetric Noyori hydrogenation is unique.

EXAMPLE 2

[0117] Scheme 3 outlines an alternate synthesis of β-hydroxyester 5using known compound 13. This alternate route toward the segment Cprecursor allows for the introduction of affinity labels andmodifications at the C4 position as shown in Scheme 3. Applying theNoyori reduction to the known unsubstituted β-keto ester 11 provides abuilding block that can be used for the modifications at C4 of theepothilones. There has only been one report so far of C4 modification onthe epothilones and this method provides a more general route ofintroducing a variety of substituents at this position. This will alsoenable a more thorough study of the structure activity relationships ofnumerous C4 substituted analogs.

[0118] Thus, the Noyori hydrogenation of β-keto ester 11 yields theknown β-hydroxyester 12, which was reported by Ali, et. al, FormalSyntheses of Cryptophycin 1 and Arenastatin A., 38 Tetrahedron Lett.,1703-1706 (1997), hereby incorporated by reference, in 97% yield (in 97%enantiomeric excess). The Frater alkylation of β-hydroxy ester 12 yieldsthe previously reported a-methyl analog 13 also previously reported byAli et al. Formal Syntheses of Cryptophycin 1 and Arenastatin A., 38Tetrahedron Lett., 1703-1706 (1997), hereby incorporated by reference,in 71% yield (98% diastereomeric excess). A second Frater alkylation ofhydroxy ester 13 gave bis-dimethyl derivative 5 in 59% yield which wasthen converted to epothilone segment C by the chemistry shown in Scheme2.

[0119] In detail, isopropylcyclohexylamine (0.71 L, 4.32 mmol, 2.16 eq.)and 3.75 mL THF were stirred together at −25° C. n-BuLi (1.64 mL, 3.6mmol, 1.8 eq.) was added dropwise over 15 minutes. The reaction mixturewas stirred at room temperature for 15 minutes and then lowered to −78°C. Compound 13 (504 mg, 2 mmol, 1 eq.) in 2.5 mL THF was added to thereaction mixture. The temperature was gradually raised to −10° C. over 4hours and then returned to −78° C. Mel (0.17 mL, 2.66 mmol, 1.33 eq.) inHMPA (0.26 mL) was added dropwise. The reaction was stirred at −78° C.for one hour and then stirred at room temperature for 16 hours. Thereaction was quenched with 4 mL 10% HCl . The mixture was then extractedfour times with CH₂Cl₂, dried over Na₂SO₄, and concentrated.Purification by column chromatography 160 g SiO₂ (hexane/EtOAc gradient)gave 312 mg (a 59% yield) of β-hydroxyester 5.

[0120] At this stage, other substituents such as benzyl, allyl and otheralkyl groups can be introduced by using other electrophiles in thesecond Frater alkylation in place of iodomethane. The novel aspect aboutthis alternate route to segment C is the ability to alter thesubstituents at the C4 position of the epothilones using theaforementioned Frater alkylation strategy. After synthesis ofβ-hydroxyester 5, the segment C can be produced following the procedureoutlined above in Scheme 2.

[0121] It should be noted that this invention makes it possible tosynthesize several analogs of this C1-C6 segment with various chainelongations at C2 and substitution at C6 positions on the epothilones.It also allows for, as mentioned before, modifications at the C4position with other groups such as aryl, heterocyclic, alkyl andbranched alkyl. These chain extensions and substitutions are illustratedby Formula C. The synthesis of these modified derivatives can beachieved utilizing chemistry exemplified in the synthesis of segment Cin Schemes 2 and 3 respectively. These modified segments can then beutilized in the total synthesis of various analogs of epothilones.

EXAMPLE 2A

[0122] The following sets forth a preferred procedure for synthesis of aspecific C1-C6 segment C precursor using the chemistry of Scheme 3Aabove.

[0123] Synthesis of Diol 13:

[0124] Ethyl-(R)-4-cyano-3-hydroxybutanoate 12 (0.100 g, 0.637 mmol) wasdissolved in ethanol (10 mL) and the temperature was lowered to 0° C.Sodium borohydride (0.024 g, 0.637 mmol 1.00 equiv) was added to theflask and the reaction was stirred for 2 hours. An additional equivalentof NaBH₄ was added after 2 hours, and a third equivalent after 2 morehours. The reaction was gradually warmed to room temperature and allowedto stir overnight. The temperature was returned to 0° C. and thereaction was quenched with a 25% acetic acid solution in ethanol. Afterstirring for one hour, the solvents were evaporated and the residualwhite solid was filtered over a cotton plug with EtOAc washing. Thecrude product was concentrated and dried overnight on high vacuum andcarried forward to the next reaction without further purification.

[0125] Synthesis of 3-hydroxynitrile 14:

[0126] To diol 13 (0.02 gm, 0.174 mmol) taken in a 50 mL flask, 40 mL ofdry benzene was added followed by (0.043 mL, 0.191 mmol) dibutyltindimethoxide and the solution heated to reflux under argon in an oil bathwith a Dean-Stark apparatus. After about 25 mL of benzene distilled overinto the sidearm, the solution was cooled and p-methoxy benzyl chloride(0.029 gm, 0.191 mmol) and tetrabutylammonium iodide (0.096 gm, 0.26mmol) were added. After stirring at room temperature overnight thereaction mixture was stirred at 60° C. for 4 hours. Cooling to roomtemperature followed by addition of aqueous ammonium chloride andextraction with ethyl acetate gave the crude product. Columnchromatography with 35% EtOAc/Hexanes gave 25 mg of product 14 (61%yield).

[0127] Synthesis of Dimethyl-hydroxynitrile 15

[0128] Diisopropylamine (0.107 mL, 0.766 mmol) was added to 5 mL of dryTHF and the solution under argon was cooled to −78° C. in a dry ice bathwith stirring. n-butyl lithium (0.51 mL, 0.71 mmol, 1.4M) was added andthe solution allowed to rise to room temperature. The solution wasstirred for 15 minutes at room temperature and cooled again to −40° C.and the β-hydroxynitrile 14 (0.04 g, 0.17 mmol) was added and graduallywarmed. After the solution reached room temperature, the mixture wasbrought to reflux using a water bath at 60° C. for 15 minutes and then0.15 mL (excess) of methyl iodide was added. The yellowish solutionturned to a white slurry in a few minutes. After 15 minutes saturatedammonium chloride was added, the aqueous layer separated and extractedthrice with EtOAc (25 mL), dried over Na₂SO₄ and concentrated. Columnchromatography with 15% EtOAc/hexane gave 0.034 g (76% yield) of 15.

[0129] Synthesis of Ketone 16:

[0130] Nitrile 15 was (0.04 gm, 0.15 mmol) dissolved in 2 mL of dry THFunder argon and stirred as EtMgBr (0.2 mL, 0.456 mmol) was added. About5 mg (catalytic amount) of CuBr.Me₂S was added and the solution wasrefluxed for 24 hours. After cooling to room temperature, a 0.06 Msolution of aqueous citric acid was added to the reaction mixture andstirring continued for 5 hrs. The solution was then extracted five timeswith EtOAc (40 mL). The combined organic layers were washed withsaturated NaHCO₃ and brine, dried over Na₂SO₄ and concentrated. Columnchromatography with 15% EtOAc/hexanes gave 25 mg (56% yield) of ketone16.

[0131] Synthesis of Diol 17:

[0132] To compound 16 (0.064 gm, 0.217 mmol) dissolved in 9 mL ofacetonitrile and 1 mL of water (0.357 gm, 0.652 mmol) 3 equivalents ofceric ammonium nitrate was added as the reaction mixture was stirred inan ice bath. The reaction mixture was allowed to warm to roomtemperature and stir for 3 hours. Solid sodium bicarbonate 0.25 gm wasadded followed by 0.25 gms of Na₂SO₃ and stirring continued overnight.All solvents were evaporated and the solid residue filtered and washedwith ethyl acetate (50 mL), dried over Na₂SO₄ and concentrated. Thecrude product obtained was purified by column chromatography to give 26mg, 70% yield of diol.

[0133] Synthesis of Ketone 18:

[0134] To diol 17 (0.026 gm, 0.15 mmol) dissolved in 4mL of dry DCM,2,6-lutidine (0.154 mL, 7.0 equivalents) followed by TBSOTf (0.205 ml, 6equivalents) were added as the reaction mixture was stirred in an icebath. The reaction was stirred overnight and 10 mL of NH₄Cl was addedfollowed by 20 mL of CHCl₃. The organic layer was separated and theaqueous layer was extracted five times, each with 25 mL of CHCl₃, driedover Na₂SO₄ and concentrated. Column chromatography with 2% ethylacetate gave 34 mg (65% yield) of product 18.

EXAMPLE 2B

[0135] This example sets forth a synthesis in accordance with Scheme 2B.The steps leading to ester 25 are specified in Scheme 2B and are similarto the steps of Schemes 3 and 2 and the supporting examples. However,the preferred synthesis from compound 25 to 26 is set forth below.

[0136] Improved Synthesis for Ketone 26

[0137] Ethyl phenyl sulfone (3.4 g, 20 mmol, 5.5 equiv) was dissolved inTHF (50 mL) and the temperature was lowered to −78° C. n-BuLi (13 mL, 18mmol, 5.0 equiv) was added dropwise to the sulfone and a pale yellowsolution formed which was stirred at −78° C. for 1.5 hours. The ester inTHF (15 mL) was added dropwise at −78° C. to the solution containingcompound 25. The bath was removed and the reaction stirred at roomtemperature for 20 hours. The reaction was quenched with 1:1 saturatedNH₄Cl and water (30 mL) and the aqueous layer was extracted five timeswith Et₂O. The organic layer was dried over Na₂SO4 and concentrated. Thecrude was then dissolved in MeOH (50 mL) and then the temperature waslowered to 0° C. NaH₂PO₄ (2.00 g, 14.4 mmol, 4.00 equiv) was addedfollowed by Na(Hg) (4.39 g). The pink suspension which developed wasstirred at 0° C. for 1 hour and 15 minutes, and 1:1 saturated NH₄Cl andwater were added followed by dilution with Et₂O. The mercury metal wasfiltered using a funnel and a plug of glass wool. The filtrate wasextracted five times with Et₂O. The combined organic layer was washedwith H₂O and brine, dried over Na₂SO4 and concentrated. Flash columnchromatography using 5% Et₂O in hexane provided 1.3 g (90%) of theketone.

EXAMPLE 3

[0138] This example illustrates the synthesis of segment B as in Scheme4. The synthesis of the C7-C11 segment B has been achieved usingpreviously reported chemistry of Lin, et al., Efficient Total Synthesesof Pumiliotoxins A and B. Applications of Iodide-Promoted IminiumIon-Alkyne Cyclization in Alkaloid Construction 118 J. Am. Chem. Soc.,9062-9072 (1996), the teachings of which are hereby incorporated byreference, and is outlined in Scheme 4. This synthesis can also be usedto introduce various chain-elongations on this segment and to introducevarious other substituents at C-8. These modifications can beillustrated by Formula IX and their synthesis can be achieved usingchemistry exemplified in the synthesis of segment B in Scheme 4. Again,these modified segments can then be utilized in the total synthesis ofvarious analogs of epothilones.

EXAMPLE 4

[0139] This example, illustrated in Scheme 5, describes the synthesis ofaldol adduct 14 followed by the aldol reaction of the segment Cprecursor, from examples 1 or 2, with segment B, from example 3. Aldoladduct 14 was then used to synthesize segment D of Scheme 5.

[0140] The connection of the two segments C and B utilizes a highlydiastereoselective aldol reaction (Scheme 5). When the C1-C6 ketonesegment C was treated with a base, namely lithium diisopropylamide andthe resultant enolate reacted with C7-C11 aldehyde segment B a singlediastereomer 14 was observed in 65% yield. The remarkablediastereselectivity is speculated to arise from a favorable nonbondinginteraction between the C10-C11 double bond and the carbonyl group ofthe aldehyde that gives rise to the desired diastereomer. Thisconnection between these two particular segments using an aldol reactionis unprecedented. After the connection has been made, the resultantsecondary alcohol will be protected as the correspondingtert-butyldimethylsilyl ether D.

[0141] In detail, diisopropylamine (60 μL, 0.45 mmol, 1 eq) dissolved in1 mL THF was cooled to −78° C. and n-BuLi (0.33 mL, 0.43 mmol, 0.95 eq)was added dropwise. After stirring at −78° C. for 15 minutes and at 0°C. for 30 minutes the reaction mixture was recooled to −78° C. Theketone (segment C) (0.184 g, 0.45 mmol, 1 eq) dissolved in 1 mL of THFwas then added dropwise. This mixture was stirred at −78° C. for 15minutes and then warmed to −40° C. over one hour. After recooling to−78° C., the aldehyde segment B (0.022 g, 0.23 mmol, 0.5 eq) dissolvedin 0.5 mL Et₂O was added dropwise over 15 minutes. After 35 minutes at−78° C., the reaction was quenched with 2 mL saturated aqueous ammoniumchloride and warmed to room temperature. The aqueous layer was extractedfive times with Et₂O and the combined organics were dried over anhydrousmagnesium sulfate. After concentration, preparative thin layerchromatography of the residue yielded 0.047 g (21% yield) of the desireddiastereomer.

[0142] Forming segment D utilizes TBS protection of adduct 14. Aldoladduct 14 (30 mg, 0.06 mmol, 1 eq.) was diluted with 1.5 mL CH₂Cl₂ andthe temperature was lowered to −78° C. 2,6-lutidine (50 μL, 0.42 mmol, 7eq.) was added dropwise followed by tert-butyl-dimethylsilyltrifluoromethanesulfonate (TBSOTf) (70 μL, 0.30 mmol, 5 eq.). Thereaction was stirred for 15 minutes and then raised to 0° C. Thereaction was complete after 3 hours and was quenched with 5 mL NH₄Cl.The mixture was extracted three times with CH₂Cl₂ and the combinedorganic layers were washed with brine, dried over MgSO₄ and thenconcentrated. Column chromatography (5% EtOAc in hexane) gave 34 mg (96%yield) of compound D.

EXAMPLE 5

[0143] This example illustrates the synthesis of a segment A precursor(the C-12-C-20 segment) and is outlined in Scheme 6. This involves newways to set the C16-C17 trisubstituted double bond and the C12-C13cis-double bond which serves as precursor to the cis-epoxide at C12-C13in the epothilones. The methodology used to introduce the thiazolemoiety draws upon zirconium-catalyzed carboalumination chemistry asdescribed by Wipf, P. and Lim, S., Rapid Carboalumination of Alkynes inthe Presence of Water, 32 Agnew. Chem., Int. Ed. Engl., 1068-1071(1993), the teachings of which are hereby incorporated by reference.Using this chemistry, a C16-C17 alkyne bond in an appropriatelyfunctionalized C13-C17 propargylic alcohol 16 is subjected tomethylalumination in the presence of zirconocene dichloride (Cp₂ZrCl₂).The chiral propargylic alcohol 16 is obtained via the asymmetricreduction of the readily available alkynyl ketone 15. A number ofmethods have been developed during the past years for theenantioselective reduction of α,β-alkynyl ketones. The resultantalkenylalane is coupled with 2-methyl-4-bromothiazole 17 in the presenceof zinc chloride under Pd(0) catalysis as described by Negishi, E.-I.,et al., in Double Metal Catalysis in the Cross-Coupling Reaction and ItsApplication to Stereo-and Regioselective Synthesis of TrisubstitutedOlefins, J. Am. Chem. Soc., 100:2254-2256 (1978) and Negishi, E.-I., inPalladium-or Nickel-Catalyzed Cross Coupling. A New Selective Method forCarbon-Carbon Bond Formation, Acc. Chem. Res., 15:340-348 (1982), theteachings of which are hereby incorporated by reference. Scheme 8illustrates the synthesis of 2-methyl-4-bromothiazole 17 which wassynthesized by adding 9.0 mL ether and n-BuLi (1.8 mL, 2.96 mmol, 1.2eq) and stirring at −78° C. for 30 minutes. The bromothiazole (0.60 g,2.47 mirol, 1 eq.) in 3.5 mL of ether was added dropwise to the n-BuLi.After stirring for one hour, MeOTf (0.56 mL, 4.94 mmol, 2 eq.) was addeddropwise and the reaction mixture was stirred for an additional 1.5hours. The reaction was then quenched with NaHCO₃ and extracted 5 timeswith ether. The combined organic layers were washed with brine, driedover Na₂SO₄, and concentrated. 262 mg (60% yield) of the2-methyl-4-bromothiazole 17 was obtained and carried on without furtherpurification.

[0144] This coupling of the alkenylalane to 2-methyl-4-bromothiazole 17permits access to the trisubstituted E-olefin 19 stereoselectivelyfollowing the protection of the alcohol 18 as the OTBS-ether.

[0145] After the introduction of the thiazole moiety, the known primaryalcohol 21(Mulzer et al., Easy Access to the Epothilone Family—Synthesisof Epothilone B, Tetrahedron Lett., 39:8633-8636 (1998) is revealed bydeprotection of the PMB ether 19 and then oxidized to C13-C20 aldehyde22, also previously reported by Mulzer, et al., Easy Access to theEpothilone Family—Synthesis of Epothilone B, Tetrahedron Lett.,39:8633-8636 (1998), the teachings of which are hereby incorporated byreference.

[0146] The zirconium-catalyzed methylalumination strategy constitutes anovel route of constructing the C16-C17 double bond and introduction ofthe thiazole ring. The novelty lies in the unprecedented use of a chiralpropargylic alcohol like 16 in the carbometalation reaction followed bythe direct introduction of the thiazole unit.

[0147] This methodology also allows for the introduction of varioussubstituents and chain elongations on the C12-C20 segment A. Thusstarting with analogs of the ketone 15 in Scheme 6, a variety ofchain-elongated derivatives of segment A can be accessed. Also carryingout an ethylalumination (Et₃Al) in place of methylalumination (AlMe₃)will allow the introduction of an ethyl group (Et) at C16. In the samecontext, other groups such as alkyl and benzyl groups can also beintroduced using the alternate stannylcupration-alkylation method byreplacing iodomethane with other electrophiles in this reaction shownbelow in Example 6 and illustrated in Scheme 7. In addition the thiazolering can be replaced by other cyclic, aromatic and heteroaromatic ringsby using other vinyl or aromatic/heteroaromatic halides in place of2-methyl-4-bromothiazole 17 in the coupling reaction following eitherthe carboalumination or stannylcupration strategy exemplified in Schemes6 and 7 respectively.

[0148] These aforementioned modifications are best illustrated byFormula E. Again all of these modified segments can then be utilized inthe total synthesis of various analogs of epothilones.

EXAMPLE 6

[0149] Scheme 7 illustrates an alternative way to introduce thetrisubstituted olefin. This method utilizes thestannylcupration-methylation methodology described by Harris, L., etal., in Synthetic Approaches to Rapamycin 3. Synthesis of a C1-C21Fragment, Synlett, 903-905 (1996), the methodology of which is herebyincorporated by reference. Thus the O-TBS ether 16a from Scheme 7 ofpropargylic alcohol 16 on treatment with the stannylcuprate reagent 20followed by methylation with iodomethane would provide the correspondingstannane which would then be coupled under Stille conditions with thebromothiazole 17 to yield the olefin 19.

EXAMPLE 7

[0150] The synthesis of 2-methyl-4-bromothiazole 17 from the known2,4-dibromothiazole is outlined in Scheme 8. This 2,4-dibromothiazolehas been previously reported by Reynaud, P., Robba, M. and Moreau, R. C.in Sur une Nouvelle Synthese du Cycle Thiazolique, Bull. Soc. Chim. Fr.,295:1735-1738 (1962), the teachings of which are hereby incorporated byreference.

EXAMPLE 8

[0151] This example illustrates the stereoselective construction of theC12-C13 cis-olefinic bond, the process of which is outlined in Scheme 9.The method shown provides maximum control over the olefin geometry aswell as furnishes common intermediates in the synthesis of bothepothilones A and B. This method is also amenable to introduction ofaffinity labels at C-12.

[0152] The C12-C13 olefin is constructed in the form of Z-vinyl iodidesA that can be obtained from vinylstannanes with defined configurations.The vinyl stannanes will be accessed by using the chemistry reported byLipshutz et al., in Preparation of Z-Vinylstannanes via Hydrozirconationof Stannylacetylenes, Tetrahedron Lett., 33:5861-5864 (1992) andLipshutz, B. H. and Keil, R. in Hydrozirconation/Transmetalation ofAcetylenic Stannanes. New 1,1-Dimetallo Reagents, Inorganica ChimicaActa, 220:41-44 (1994), the teachings of which are hereby incorporatedby reference. The chemistry utilizes a 1,1-dimetallo species as astereodefined 1,1-vinyl dianion synthon.

[0153] The synthesis starts with a Corey-Fuchs reaction (PPh₃, CBr4), asdescribed by Corey, E. J. and Fuchs, P. L., A Synthetic Method forFormyl to Ethynyl Conversion, Tetrahedron Lett., 36:3769-3772 (1972) ofthe known aldehyde 22, followed by base-induced elimination andquenching of the lithium acetylide with tributyltin chloride (Bu₃SnCl)to yield alkynylstannane 23. The 1,1-dimetallo species 24 is generatedby hydrozirconation of the alkynyl stannane 23 usingchlorohydridozirconocene (Schwartz reagent). An aqueous quench providesZ-vinylstannane 25a or alternatively, selective transmetalation with ahigher order cuprate, followed by addition of an electrophile (MeOTf incase of epothilone B) to the resultant species provides theα-substituted vinylstannane 25b with high stereoselectivity. TheZ-vinylstannanes 25a and 25b can then be transformed to thecorresponding vinyl iodides A utilizing iodine with retention ofconfiguration.

[0154] In summary, although the vinyl iodides A are previously reportedcompounds as evidenced by the teachings of Schinzer, D. et al., in TotalSynthesis of (−)-Epothilone A, 36 Angew. Chem., Int. Ed. Engl., 523-524(1997) and Schinzer, D., Bauer, A., and Scheiber, J., Synthesis ofEpothilones: Stereoselective Routes to Epothilone B, Synlett, 861-864(1998), the teachings of both are hereby incorporated by reference, themethod to synthesize it from the known aldehyde 22 is different fromconditions reported in other total syntheses of epothilones. Inaddition, the above mentioned hydrozirconation reactions providesprecise control over the geometry of the C12-C13 olefin bond and thismethodology constitutes a unique way to construct this double bond. Alsothe use of other electrophiles in the transmetalation reaction with theintermediate species 24 allows for the synthesis of various analogsmodified at C12 position of the epothilones as illustrated in Formula E.

EXAMPLE 8A

[0155] This example illustrates the important steps in the chemistrydepicted in Scheme 9B, for the production of vinyl halide C12-C20precursors.

[0156] Synthesis of Enone 3:

[0157] Barium hydroxide octahydrate (0.21 g, 0.68 mmol) was added to thebeta-ketophosphonate 2 dissolved in 2.5 mL of anhydrous THF. After 45minutes at room temperature, this mixture was cooled to 0° C. and thealdehyde 1 dissolved in 2.4 mL of THF and 0.12 mL of water was addeddropwise. The reaction mixture was stirred at 0° C. for one hour andthen warmed to room temperature over 45 minutes. The mixture was dilutedwith 10 mL of dichloromethane and 10 mL of saturated aqueous sodiumbicarbonate was then added. The aqueous layer was extracted thrice withdichloromethane (5 mL) and combined organics dried over anhydrousmagnesium sulfate. Flash column chromatography of the crude materialafter concentration on silica gel using a gradient of hexanes anddiethyl ether (5% ether-hexanes to 30% ether-hexanes) gave 208 mg of theenone (75% yield).

[0158] Synthesis of Alcohol 4:

[0159] (R)-Me-CBS-oxazaborolidine (0.08 mL, 0.08 mmol, 1M solution intoluene) was placed in a 10 mL flask and toluene was removed in vacuo.The residual solid was dissolved in 0.3 mL of dichloromethane and boranedimethylsulfide complex (0.23 mL, 0.23 mmol, 1M in dichloromethane) wasadded dropwise. This mixture was cooled to 0° C. and the enone 3 (0.05g, 0.154 mmol) dissolved in 0.3 mL dichloromethane was added dropwiseover a period of one hour. After stirring at 0° C. for two hours,methanol (0.5 mL) was carefully added followed by 1,2-ethanolamine (0.3mL). The reaction mixture was stirred for another 16 hours at roomtemperature following which it was poured into 15 mL of ethyl acetateand washed with saturated aqueous ammonium chloride, water and brine.The organic layer was then dried over anhydrous magnesium sulfate,concentrated and the crude was then purified by flash columnchromatography on silica gel using 70:30 hexanes:diethyl ether to 50:50hexanes:diethyl ether solvent mixture as eluent. The desired alcohol wasobtained in 70% yield (35 mg) and in 95% enantiomeric excess.

[0160] Synthesis of Bis-silylether 5:

[0161] The alcohol 4 (0.24 g, 0.73 mmol) dissolved in 5 mL ofdichloromethane was cooled to −78° C. and 2,6-lutidine (0.13 mL, 1.095mmol) was added. TBSOTf(0.2 mL, 0.88 mmol) was then added. Afterstirring for 15 minutes, the reaction mixture was quenched withsaturated aqueous ammonium chloride (5 mL) and then the aqueous layerwas extracted thrice with dichloromethane. The combined organics weredried over anhydrous magnesium sulfate. Flash column chromatography ofthe crude after concentration using silica gel and 13:1 hexanes: diethylether as eluent provided 0.257 g (80% yield) of the known bis-silylether 5.

Example 8B

[0162] This example illustrates the chemistry depicted in Scheme 9C,using the known aldehyde 6 as a starting material to produce the desiredvinyl halide precursor.

[0163] Synthesis of Alcohol 7:

[0164] The enolate of ethyl acetate (1.76 mL, 18.0 mmol, 1.00 equiv) wasgenerated at −78° C. using LDA (19.8 mmol, 1.10 equiv). The knownaldehyde 6 was added dropwise and the reaction mixture turned brightorange. After 10 minutes the reaction was quenched with NH₄Cl (20 mL).Brine (20 mL) was added and the aqueous layer was extracted four timeswith Et₂O (50 mL). The combined organic layer was dried over MgSO₄ andconcentrated. Column chromatography (hexane/ether gradient 1: 1, 3:1,100% Et₂O) gave 3.5 grams (76% yield) of the desired alcohol 7.

[0165] Synthesis of Beta-ketoester 8:

[0166] MnO₂ (23.4 grams, 10 wt %) was placed in dry chloroform (40 mL)and the temperature was lowered to 0° C. The alcohol 7 (2.34 grams, 1equiv) was added to the MnO₂ suspension in chloroform (25 mL). Thereaction was warmed to room temperature and stirred for 17 hours. Themixture was then filtered through Celite which was rinsed four timeswith CH₂Cl₂ (50 mL). The combined organic layer was dried (Na₂SO₄),concentrated, and passed through a short column with 100% Et₂O toprovide 1.95 g (90%) of beta-ketoester 8.

[0167] Synthesis of Chiral Alcohol 9:

[0168] Acetone and methanol were each degassed 5 times using thefreeze-thaw method. (S)-BINAP (74 mg, 0.12 mmol, 15 mol%) and theruthenium complex (38 mg, 0.12 mmol, 15 mol%) were combined in atwo-neck flask with acetone (9 mL) and HBr solution (0.85 mL: 5.1 mLacetone and 0.25 mL 48% HBr). This mixture was stirred at roomtemperature for two hours, after which the acetone was removed underreduced pressure. The catalyst was transferred with MeOH (4 mL total)into a Parr flask containing the beta-ketoester 8 in MeOH (4 mL) total.(Beta-ketoester 8 in MeOH, 2 mL, was deoxygenated three times prior totransfer to Parr flask). The hydrogenation reaction was carried out fortwo hours at 52.5 psi and at room temperature. The MeOH was removed andthe residue was redissolved in ether and filtered. Column chromatographyin (1:1 hexane/Et₂O) provided 100 mg (50% yield) of the desired alcohol9. The enantiomeric excess was determined to be 83% by chiral HPLC (95:5hexane/EtOH, 254 nm, 1 mL/min, Chiracel OD—H).

[0169] Synthesis of Silyl Ether 10:

[0170] The alcohol 9 (267 mg, 1.05 mmol, 1.00 equiv) was dissolved inCH₂Cl₂ (5 mL), and the temperature was lowered to 0° C. 2,6-lutidine(0.260 mL, 2.23 mmol, 2.13 equiv) was added followed by TBSOTf (0.375 L,1.63 mmol, 1.55 equiv). The reaction was stirred for 1.5 hours and thenquenched with water (2 mL). The mixture was extracted four times withCH₂Cl₂ and the combined organic layer was washed with brine, dried overNa₂SO₄, and concentrated. Column chromatography (3:1 hexane/Et₂O) gave276 mg (72% yield) of the protected ester 10.

[0171] Synthesis of Primary Alcohol 11:

[0172] The protected ester (275 mg, 1.03 mmol, 1.00 equiv )was dissolvedin CH₂Cl₂ (5 mL) and the temperature was lowered to −78° C. DIBAL-H (5mmol, 1.0 M in hexanes) was added dropwise and after 6 hours, thereaction was quenched with saturated potassium sodium tartrate (15 mL)and allowed to stir two hours until the phases had separated. Themixture was then extracted four times with CH₂Cl₂. The combined organiclayers were dried over sodium sulfate (Na₂SO₄) and concentrated. Aftercolumn chromatography (hexane/ether gradient 1:1, 3:1, 100% Et₂O), 80 mgof known alcohol 11 was obtained.

EXAMPLE 9

[0173] This example and the next (Example 10) illustrate C11-C12 bondconstruction including coupling of the C1-C11 (segment D) and C12-C20(segment A) subunits and completion of the total synthesis.

[0174] Having defined all the requisite stereocenters and geometries,the stage is set for the union of the C1-C11 and the C12-C20 subunits.The C11-C12 bond connection is achieved by the B-alkyl Suzuki reactionof the C1-C11 olefin D with the vinyl iodides A shown in Scheme 10 toafford the precursors 26 for the synthesis of epothilone A and B. ThisB-alkyl Suzuki reaction is described by Miyaura, N., and Suzuki, A. inPalladium-Catalyzed Cross-Coupling Reactions of Organoboron Compounds,Chem. Rev. 95:2457-2483 (1995), the teachings of which are herebyincorporated by reference. Thus, hydroboration of the olefin D with9-BBN followed by coupling of the corresponding organoborane with thevinyl iodides A in presence of [PdCl₂(dppf)₂] would yield the olefin 26.

EXAMPLE 10

[0175] Finally the conversion of the coupled C1-C20 products 26 toepothilones A and B is accomplished using the previously reportedprocedures of Nicolaou, K. C. et al., in Total Syntheses of EpothilonesA and B via a Macrolactonization-Based Strategy J. Am. Chem. Soc.,119:7974-7991 (1997), the teachings of which are hereby incorporated byreference. Thus selective deprotection at C I by camphorsulfonic acid(CSA), shown in Scheme 11, followed by sequential oxidation of theprimary alcohol first under Swem conditions followed byNaClO₂—NaH₂PO₄furnishes the known acids. Selective deprotection of theTBS ether at C15 using tetrabutylammonium fluoride yields the hydroxyacids 27. The key macrolactonization step is then carried out using theYamaguchi method as descibed by Inanaga, J. et al., in A RapidEsterification by Means of Mixed Anhydride and its Application toLarge-ring Macrolactonization, Bull. Chem. Soc. Jpn., 52:1989-1993(1979), the teachings of which are hereby incorporated by reference,affording the known 16-membered macrolides 28. The final stages in thesynthesis involve the deprotection of both the TBS groups from themacrolides 28 (TFA, CH₂Cl₂) and the diastereoselective epoxidation ofthe C12-C13 double bond with epoxidizing agents such asdimethyldioxirane or methyl(trifluoromethyl)dioxirane.

[0176] As shown in Formulae I, IX and X, various modified segments canbe employed during the syntheses of the individual segments (C, B andA). All of these modified segments can then be connected using the bondconnections (Aldol reaction and the B-alkyl Suzuki reaction) highlightedin this total synthesis. This would provide numerous homologs, analogsand affinity labels of the epothilones. All references noted herein areexpressly incorporated by reference.

[0177] Acronym and Symbol Definitions

[0178] In order to facilitate the preceding discussion various acronymsand symbols have been used. These have the following definitions. Acacetyl Ar aromatic 9-BBN 9-borabicyclo[3.3.1]nonane (S)-BINAP(S)-(-)-1,1′bi-2-naphthal(s)-(-)-2,2′-Bis(diphenylphos-phino)1,1′binaphthyl Bn benzyl BnO benzyloxy Bu butyl n-BuLi n-butyllithium Cp₂ZrCl₂ zirconocene dichloride Cp₂Zr(H)Clchlorohydridozirconocene CSA camphorsulfonic acid DDQ2,3-dichloro-5,6-dicyano-1,4-benzoquinone DIBAL-H diisobutylaluminumhydride DIEA diisopropylethylamine (+)DIPT diisopropyl tartrate DMFN,N-dimethylformamide DMPU1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone DMSO dimethylsulfoxideee enantomeric excess Et ethyl HBTU O-(benzotriazol-1-y-1)-N,N,N′,N′tetramethyluronium hexafluoro phosphate HF hydrogen fluoride HMPAhexamethylphosphoramide LDA lithium diisopropylamide LHMDS lithiumhexamethyldisilazide LICA lithium isopropylcyclohexylamide m-CPBAmeta-chloroperoxybenzoic acid MDR multi-drug resistant Me methyl MeOTfmethyl triflate MS molecular sieves MsCl mesyl chloride NaHMDS sodiumhexamethyldisilazide NMO 4-methylmorpholine N-oxide OTf trifluoromethanesulfonate PdCl₂(dppf)₂ dichloro[1,1′-Bis(diphenylphosphino)ferrocene]palladium II Pd(PPh₃)₄ tetrakis (triphenylphosphine) palladium (0) Phphenyl PPh₃ triphenylphosphine PMB para-methoxybenzyl Ru cat bis-(2methylallyl) cycloocta-1,5-diene ruthenium (II) TBAF tetrabutylammoniumfluoride TBHP tertbutyl hydroperoxide TBS tertiary-butyldimethylsilylTBSCl tertiary-butyldimethylsilyl chloride TBSOTftertiary-butyldimethylsilyl triflate TFA trifluoroacetic acid THFtetrahydrofuran Ti(O-iPr)₄ titanium isopropoxide TPAPtetrapropylammonium perruthenate

We claim:
 1. A method of synthesizing in high enantomeric excess analcohol of the formula

where R₈ is selected from the group consisting of H, C1-C4 straight orbranched chain alkyl, alkenyl or alkynyl groups, R₉ is selected from thegroup consisting of H, C1-C10 straight and branched chain alkyl,alkenyl, alkynyl, hydroxyalkyl, hydroxyalkenyl and hydroxyalkynylgroups, substituted and unsubstituted cyclic, heterocyclic and arylgroups, X₂ is O or S, P′ is a protective group, and n₄ is an integerwhich ranges from 1 to 4, said method comprising the step of: providingan enone compound of the formula

 where R₈, R₉, X₂, P′ and n₄ are as defined above; and asymmetricallyreducing said enone compound in the presence of a chiral catalyst toobtain said alcohol.
 2. The method of claim 1, including the step ofproviding said enone compound by reacting in a basic reactive medium analdehyde compound of the formula

where R₉ and X₂ are as defined in claim 1, with a phosphonate compoundof the formula

 where R₈, P′ and n₄ are as defined in claim 1, R₁₀ and R₁₁ areindividually selected from the group consisting of C1-C4 straight orbranched chain alkyl groups.
 3. The method of claim 1, wherein R₈ and R₉are each H, X is S, n is 1, and P′ is TBS.
 4. The method of claim 1,said asymmetric reduction reaction being carried out at a temperature offrom about −20 to 40° C.
 5. The method of claim 1, said chiral ispreferably (R)—B—Me—CBS-oxazaborolidine.
 6. A method of synthesizing theC12-C20 epothilone precursor of the formula

where R8 is selected from the group consisting of H, C1-C4 straight orbranched chain alkyl, alkenyl or alkynyl groups, R₉ is selected from thegroup consisting of H, C1-C10 straight and branched chain alkyl,alkenyl, alkynyl, hydroxyalkyl, hydroxyalkenyl or hydroxyalkynyl groups,substituted and unsubstituted cyclic, heteroxylic and aryl groups, R₁₂is selected from the group consisting of H, C1-C10 straight and branchedchain alkyl groups, substituted and unsubstituted benzyl groups, andC1-C10 alkoxy groups, X₂ is O or S, n is an integer which ranges from 1to 4, P′ is a protective group and M is either iodine or bromine,wherein an alcohol of the formula

where R₈, R₉, X₂, n₄ are as defined above and P′ is a protective group,is converted to the C12-C20 epothilone segment A, which method comprisesthe steps of: providing an enone compound of the formula

 where R₈, R₉, X₂, P′ and n₄ are as defined above; and asymmetricallyreducing said enone compound in the presence of a chiral catalyst toobtain said alcohol.
 7. The method of claim 6, including the step ofproviding said enone compound by reacting in a basic reactive medium analdehyde compound of the formula

where R₉ and X₂ are as defined in claim 6, with a phosphonate compoundof the formula

 where R₈, P′ and n₄ are as defined in claim 6, and R₁₀ and R₁₁ areindividually and respectively selected from the group consisting ofC1-C4 straight or branched chain alkyl groups.
 8. The method of claim 6,wherein R₈ and R₉ are each H, X is S, n₄ is 1, and P′ is TBS.
 9. Themethod of claim 6, said chiral catalyst is preferably(R)—B—Me—CBS-oxazaborolidine.
 10. A method of synthesizing the C12-C20epothilone precursor of the formula

where R₈ is selected from the group consisting of H, C1-C4 straight orbranched chain alkyl, alkenyl or alkynl groups, R₉, is selected from thegroup consisting of H, C1-C10 straight and branched chain alkyl,alkenyl, alkynl, hydroxyalkyl, hydroxyalkenyl and hydroxyalkynl groups,substituted and unsubstituted cyclic, heteroxylic and aryl groups, R₁₂is selected from the group consisting of H, C1-C10 straight and branchedchain alkyl groups, substituted and unsubstituted benzyl groups, andC1-C10 alkoxy groups, X₂ is O or S, n is an integer which ranges from 1to 4, P′ is a protective group and M is either bromine or iodine, saidmethod comprising the steps of: providing an aldehyde of the formula

 where R₈, R₉, and X₂ are as defined above; reacting said aldehyde withan acetate of the formula

where R₁₃ is a C1-C4 alkyl group, Z is a C1-C4 straight or branchedchain alkyl group or a substituted or unsubstituted benzyl group in abasic reaction mixture to yield a β-hydroxyester of the formula

 where R₈, R₉, X₂, Z and n4 are as defined above; oxidizing saidβ-hydroxyester to the corresponding β-ketoester of the formula

 where R₈, R₉, X₂, Z and n4 are as defined above; hydrogenating saidβ-ketoester to form a chiral alcohol of the formula

where R₈, R₉, X₂, Z and n₄ are as defined above, by reacting theβ-ketoester with a hydrogenating agent in the presence of asymmetricorganometallic molecular catalyst comprising a metal atom or ion havingone or more chiral ligands coupled thereto; and converting said chiralalcohol to said C12-C20 epothilone precursor.
 11. The method of claim10, said acetate being ethyl acetate.
 12. The method of claim 10,wherein said aldehyde and acetate are reacted in the presence of analkali metal diisopropyl amide in a solvent selected from the groupconsisting of THF, a mixture of t-butanol and t-butoxide, sodiumethoxide, and ethanol.
 13. The method of claim 10, wherein said aldehydeand acetate are reacted at a temperature of from about −50 to −125° C.14. The method of claim 10, wherein said β-hydroxyester is oxidizedusing an alkali metal or alkaline earth metal oxide or hydroxide. 15.The method of claim 10, wherein said hydrogenating agent is hydrogen.16. The method of claim 10, said hydrogenating step being carried out ata pressure of from about 30-100 psi.
 17. The method of claim 10, saidhydrogenating step being carried out at a temperature of from about40-100° C.
 18. A method of synthesizing a C1-C6 epothilone precursor ofthe formula

where n₁ is an integer from 0-4, R₄ is selected from the groupconsisting of H, C1-C10 straight and branched chain alkyl groups,substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups,R₅ and R₆ are each individually and respectively selected from the groupconsisting of H, substituted and unsubstituted aryl and heterocyclicgroups, C1-C10 straight and branched chain alkyl groups, and substitutedand unsubstituted benzyl groups, R₇ is H or straight or branched chainC1-C10 alkyl groups, and P′ is a protective group, comprising the stepsof: providing a nitrile compound of the formula

where P′, R₅, R₆, R₇ and n₁, are as defined above, and the value of eachn₁, may be the same or different; alkylating said nitrile compound toyield a dialkylated compound of the formula

where P′, R₅, R₆, R₇, and n₁, are as defined above, and the value ofeach n₁, may be the same or different; and converting said nitrilecompound to yield said epothilone precursor.
 19. The method of claim 18,said converting step comprising the steps of oxidizing said dialkyatedcompound to yield a ketone of the formula

where P′, R₄, R₅, R₆, and n₁ are as defined in claim 18; and convertingsaid ketone to said C1-C6 epothilone precursor.
 20. The method of claim18, said converting step comprising the steps of deprotecting saidnitrile compound to yield a diol compound having the formula

where R₄, R₅, R₆, n₁ are as defined in claim 18, and thereafterconverting said diol compound to said C1-C6 epothilone precursor.
 21. Amethod of synthesizing a C1-C6 epothilone precursor of the formula

where n₁ is an integer from 0-4, R₄ is selected from the groupconsisting of H, C1-C10 straight and branched chain alkyl groups,substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups,R₅ and R₆ are each individually and respectively selected from the groupconsisting of H, substituted and unsubstituted aryl and heterocyclicgroups, C1-C10 straight and branched chain alkyl groups, and substitutedand unsubstituted benzyl groups, R₇ is H or straight or branched chainC1-C10 alkyl groups, and P′ is a protective group, said methodcomprising the steps of: providing an ester compound of the formula

 where R₁, R₅, R₆, R₇, n₁ and P′ are as defined above, and R′ is aC1-C10 striaght or branched chain alkyl group; reacting said estercompound with a sulfone to acylate the ester, and thereafterdesulfonating the acylated ester to obtain said epothilone precursor.22. The method of claim 21, said sulfone being of the formulaX₁—SO₂—CH₂R₄ where X₁ is selected from the group consisting of C1-C10straight and branched chain alkyl, alkenyl, and alkynyl groups, andsubstituted and unsubstituted aryl and heterocyclic groups, and R₄ is asdefined above.
 23. The method of claim 22, said sulfone being ethylphenyl sulfone.